HPLC Separation of 2-Ethyl-5(6)-methylpyrazine and Its Electroantennogram and Alarm Activities on Fire Ants (Solenopsis invicta Buren)

Abstract

2-Ethyl-3,6-dimethylpyrazine (EDMP) was an alarm pheromone component isolated from the mandibular gland of the red imported fire ant, Solenopsis invicta Buren. Several pyrazine analogues have been previously found to elicit significant alarm responses in S. invicta workers. This study aimed to separate the commercially available 2-ethyl-5(6)-methylpyrazine (EMP), i.e., a mixture of 2-ethyl-6-methylpyrazine (2E6MP) and 2-ethyl-5-methylpyrazine (2E5MP), and to examine both electroantennogram (EAG) and behavioral responses of S. invicta workers to EMP and the purified isomers. HPLC separations were achieved using a polysaccharide chiral stationary phase (Chiralpak AD-H) column with both mobile phases: Cyclohexane/isopropanol, and hexane/isopropanol. A ratio of 99:1 was selected for the separation of EMP at semipreparative level. The structures of the isomers obtained through the cyclohexane/isopropanol mobile phase were confirmed by detailed analyses of 2D-HSQC- and -HMBC-NMR data. The two isomers showed differential methine C–H correlations evidenced by 2D-HMBC-NMR spectra. The two concentrated fractions obtained through hexane/isopropanol mobile phase were subjected to EAG test and behavioral bioassay on S. invicta workers. The two HPLC−purified isomers, 2E6MP and 2E5MP, and their mixture (1:1) at same dose elicited similar EAG and alarm responses, indicating that these two isomers are equally active. The 2D-NMR−spectroscopic characterization, and electrophysiological and alarm activities of 2E6MP and 2E5MP were reported here for the first time.

An evolutionary conserved olfactory receptor for foodborne and semiochemical alkylpyrazines

“…[7][8][9][10][11] "Semiochemicals" are mostly, but not exclusively, volatile compounds that allow the transfer of chemical cues between individuals of the same and/or different species, most often eliciting a standardized behavior. [12][13][14][15] Pyrazines function both intraspecies, for example, as pheromones sensu stricto, [16][17][18] as well as interspecies, for example, as allomones (to the benefit of the "sender") 7,8 and kairomones (to the benefit of the "receiver"). 9,10,19 Despite pyrazines' eminent role as information carriers for the chemical senses of humans and other animals, the molecular chemoreceptive mechanisms of their chemosensory perception remained largely unknown, so far.…”

Electrophysiological and Alarm Responses of Solenopsis invicta Buren (Hymenoptera: Formicidae) to 2-Ethyl-3,5-dimethylpyrazine

“…Although perception of alarm pheromone has an important role in the chemical communication in ants, only few studies have tested EAG responses of ants to their alarm pheromones and related analogs [18,23,24,26,29,30]. EAG responses in fire ant workers under the treatment of 2-ethyl-3,5-dimeth ylpyrazine ranged from 0.06 to 1.56 mV (Figure 2).…”

“…The antennal sensitivity of S. invicta workers to 2-ethyl-3,5-dimethylpyrazine was evaluated by conventional EAG methods as described in previous studies [18,24,26]. Briefly, an isolated ant head with antennae was connected between two glass electrodes filled with Ringer's solution modified by Tween ® 80 [27].…”

Semibiocatalytic Approach toward Regioisomerically Enriched Ethyl Dimethylpyrazines Important in Flavor Industries

“…The EDMP isomeric mixture (Sigma-Aldrich, St. Louis, MO) contained a 50:50 mixture of each isomer (235-EDMP and 325-EDMP), as determined by GC/MS analysis. Guided by earlier high-performance liquid chromatography (HPLC) solvent systems to separate pyrazines by C-18 RP-HPLC, ,, various isocratic mixtures of CH 3 CN (20–50%) in water were tested as the eluent to optimize the resolution of elution ( R S ). A 1 μL aliquot from a 37 μM (5 mg EDMP/mL CH 3 CN) solution was loaded onto the analytical C18 column (Alltech, 250 mm × 2.1 mm i.d., 5 μm particle size) to determine that a solvent mixture of CH 3 CN/water (20:80, isocratic) was suitable as the mobile phase for a semipreparative column.…”

“…The EDMP isomers were quantified using a standard curve with 2,5-DMP as the internal standard. The converted yields 33,48,49 various isocratic mixtures of CH 3 CN (20−50%) in water were tested as the eluent to optimize the resolution of elution (R S ). A 1 μL aliquot from a 37 μM (5 mg EDMP/mL CH 3 CN) solution was loaded onto the analytical C18 column (Alltech, 250 mm × 2.1 mm i.d., 5 μm particle size) to determine that a solvent mixture of CH 3 CN/water (20:80, isocratic) was suitable as the mobile phase for a semipreparative column.…”